Human induced pluripotent stem cells for high efficiency genetic engineering

ABSTRACT

Methods are disclosed herein for efficiently generating human induced pluripotent stem cells (iPSC) containing a nucleic acid including a doxycycline promoter operably linked to a nucleic acid encoding Cas9. These methods include transfecting a human somatic cell with a nucleic acid molecule comprising a doxycycline promoter operably linked to a nucleic acid encoding a Cas9, and constitutive promoter operably linked to a tetracycline responsive element and inducing the somatic cell to form an iPSC, thereby producing an iPSC that can undergo CRISPR/Cas9-mediated recombination at a high efficiency. The human iPSC, or a cell differentiated therefrom, is cultured in the presence of doxycycline to induce expression of the Cas9. These cells can then be used to target in any gene of interest by introducing nucleic acids encoding sgRNAs. Induced pluripotent stem cells produced by these methods are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION(S)

This claims the benefit of U.S. Application No. 62/369,698, filed Aug.1, 2016, which is incorporated herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant no. DK099257awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

This relates to the field of stem cells, specifically for methods ofgenerating induced pluripotent stem cells containing Cas9, and allowinghigh efficiency that can be genetic engineering.

BACKGROUND

Patient-specific induced pluripotent stem cells (iPSCs) derived fromsomatic cells provide a unique tool for the study of human disease, andare a promising source for cell replacement therapies. One cruciallimitation has been the inability to perform experiments undergenetically defined conditions. This is particularly relevant for lateage onset disorders in which in vitro phenotypes are predicted to besubtly susceptible to significant effects of genetic backgroundvariations combined with epigenetic alterations. Moreover, there is aclear need for effective therapy strategies for a number of chronicdiseases with genetic and epigenetic backgrounds (e.g., nonalcoholicsteatohepatitis, alcohol-induced liver disease, aging, Parkinson'sdisease, heart failure), that require a deep understanding of themechanisms responsible for the disease's evolution to organ/celldysfunction in human tissue. Available animal models for these diseaseshave been extremely useful for elucidating many aspects of thedisorders, but the relative roles of the pathways in humans have notbeen conclusively determined. Most simply stated, mice are not men.

Disclosed herein are high efficiency methods for genome editing iniPSCs. By combining approaches involving genome editing and iPSCtechnology, generally applicable solutions are provided for addressingsuch problems by generating sets of isogenic disease and control humanpluripotent stem cells.

SUMMARY

Methods are disclosed herein for generating a human induced pluripotentstem cells. The methods include transfecting a human somatic cell with anucleic acid molecule comprising a doxycycline promoter operably linkedto a nucleic acid encoding a Cas9, and constitutive promoter operablylinked to a tetracycline responsive element, and inducing the somaticcell to form an induced pluripotent cell. These methods produce inducedpluripotent stem cells that can undergo CRISPR/Cas9-mediatedrecombination at a high efficiency, wherein the human inducedpluripotent cells or cells differentiated therefrom are cultured in thepresence of doxycycline to induce expression of the Cas9. In someembodiments, the cells are human.

In further embodiments, these cells used to target in any gene ofinterest by introducing nucleic acids encoding sgRNAs.

Induced pluripotent stem cells produced by these method are alsodisclosed.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. FIG. 1A is a schematic of the pcLVI(3G) vector used inconditional systems. FIG. 1B is a schematic diagram ofpCLVi(3G)-Tet-ON-3G. The Tet-on-3G system is composed of these twoelements: (1) a reverse tetracycline-controlled transactivator induciblepromoter (rtTA-3G) expressed constitutively, under the control of anUbiquitin C promoter; and (2) a Tetracycline Response Element (pTRE-3G)controlling the transcription of a sequence of interest. The pTRE-3G iscomposed of 7 repeats of the 19 bp bacterial tet-O sequence placeupstream of a minimal promoter with very low basal expression in theabsence of Tet-On. The rtTA-3G protein binds the pTRE-3G only if boundby a doxycycline. The addition of doxycycline to the system initiatesthe transcription of the sequence of interest.

FIG. 2. Bright field microscopy of transduced and non-transduced hFFafter 7 days of puromycin selection.

FIG. 3. Puromycin resistance gene expression in transduced andnon-transduced hFF assessed by means of qRT-PCR.

FIG. 4. Bright field and live florescence microscopy of hFF withfluorescent RFP and GFP markers 48 h after doxycycline induction.

FIG. 5. Cas9 complex #1 and #2 in hFF-TET-ON-Cas9 with and withoutaddition of doxycycline for 48 h assessed by means of RTqPCR.

FIG. 6. Bright Field and live fluorescence microscopy ofhFF-TET-ON-TagRFP reprogramming into hiPS-TET-ON-TagRFP. hFF transducedwith GFP be followed up to 14 days after transduction. Clones started toappear at day 15.

FIG. 7. Bright field microscopy hiPS-TET-ON-TagRFP and hiPS-negativecontrol after 24 h of puromycin selection.

FIG. 8. Fluorescence microscopy of hiPS-TET-ON-TagRFP 48 h afterdoxycyclin exposure. Nuclei were counterstained with DAPI.

FIG. 9. Puromycin resistance gene expression in hiPS-TET-ON-TagRFPclones assessed by means of qRT-PCR.

FIG. 10. Summary table of hiPS-TET-ON-RFP generation and efficiency ofinducible system.

FIGS. 11A-11B. Characterization of hiPS-TET-ON-TagRFP cells. A)Immunofluorescence of Nanog, Oct3.4, TRA-1-60, SSEA4 inhiPS-TET-ON-TagRFP. Nuclei were counterstained with DAPI. B) Oct3/4,Lin28 and C-myc expression in ES and hiPS-TET-ON-TagRFP cells assessedby means of RTqPCR.

FIGS. 12A-12B. Generation of embryoid bodies with hiPS-TET-ON-TagRFP. A)Bright Field microscopy of hIPS-TET-ON-TagRFP 15 days after embryoidbodies formation. B) Immunofluorescence of the three germ layers GATA-4and SOX17 for Endoderm, HAND1 and Brachyury for Mesoderm and Otx-2 andSOX1 for Ectoderm on hiPS-TET-ON-TagRFP. Nuclei were counterstained withDAPI.

FIG. 13. Schematic of methods for generation of hiPS-Tet-On-Cas9systems.

FIGS. 14A-14B. Generation and characterization of hiPS-Cas9/GFP.Doxycycline inducible human iPS cells design to carry a Cas9 systemspecifically for gain of function experiments and a GFP reporter.hiPS-Cas9/GFP displays high expression Cas9 expression and GFP into 100%of cells (A). When nucleofected with sgRNA for EGFR or HNF4 promoters(B), hiPS-Cas9/GFP show a strong increase of either EGFR or HNF4 whenCas9 system is activated, validating high CRISPR/Cas9 activity level ofthis cell line. Scale bar: 50 um. Data are presented as mean −/+SEM,with P<0.05.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [Sequence_Listing, Jul. 31, 2017, 16,471 bytes], which isincorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary nucleic acid sequence of a doxycyclinepromoter.

SEQ ID NO: 2 is an exemplary amino acid sequence of a Streptococcuspyogenes Cas9.

SEQ ID NO: 3 is an exemplary nucleic acid sequence of a polynucleotideencoding a tracrRNA.

SEQ ID NO: 4 is an exemplary nucleic acid sequence of a U6 promoter.

SEQ ID NO: 5 is a nucleic acid sequence of a polynucleotide encoding asgRNA.

SEQ ID NO: 6 is an exemplary nucleic acid sequence of a ubiquitinpromoter.

SEQ ID Nos: 7-8 are nucleic acid sequences of a polynucleotides encodingsgRNAs.

SEQ ID NOs: 9-10 are nucleic acid sequences of primers.

DETAILED DESCRIPTION

By combining approach-mediated genome editing and iPSC technology, agenerally applicable solution is provided to generate sets of isogenicdisease and control human pluripotent stem cells. A schematic approachto generating isogenic disease and custom-engineered pluripotent stemcells has been developed and has involved complex and low efficientmethods, as, for example in, FIG. 13.

High efficiency methods are disclosed herein for the generation ofiPSCs, such as human iPSCs. Disclosed are custom engineered-systems thatelucidate the role of transcriptional programs in the development ofhuman disease, at a single and genome wide level. An exemplary protocolis shown in FIG. 2. The robust capability to genetically modifydisease-causing point mutations in patient-derived human iPSCsrepresents a significant advancement for basic biomedical research andan advance toward hiPSC-based cell replacement therapies. Thus, providedis a generally applicable solution to a key problem, and a demonstrationof the generation of a panel of isogenic mutant and control cell linesfrom hiPSCs.

Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a cell”includes single or plural cells and is considered equivalent to thephrase “comprising at least one cell.” The term “or” refers to a singleelement of stated alternative elements or a combination of two or moreelements, unless the context clearly indicates otherwise. As usedherein, “comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.Dates of GENBANK® Accession Nos. referred to herein are the sequencesavailable at least as early as Sep. 16, 2015. All references, patentapplications and publications, and GENBANK® Accession numbers citedherein are incorporated by reference. In order to facilitate review ofthe various embodiments of the disclosure, the following explanations ofspecific terms are provided:

Alter: A change in an effective amount of a substance or parameter ofinterest, such as a polynucleotide, polypeptide or a property of a cell.An alteration in polypeptide or polynucleotide or enzymatic activity canaffect a physiological property of a cell, such as the differentiation,proliferation, or senescence of the cell. The amount of the substancecan be changed by a difference in the amount of the substance produced,by a difference in the amount of the substance that has a desiredfunction, or by a difference in the activation of the substance. Thechange can be an increase or a decrease. The alteration can be in vivoor in vitro. In several embodiments, altering is at least about a 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% increase ordecrease in the effective amount (level) of a substance, theproliferation and/or survival of a cells, or the activity of a protein,such as an enzyme.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Cell Culture: Cells grown under controlled condition. A primary cellculture is a culture of cells, tissues or organs taken directly from anorganism and before the first subculture. Cells are expanded in culturewhen they are placed in a growth medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is typically measured by the amount of time required forthe cells to double in number, otherwise known as the doubling time.

Clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (Cas9): An RNA-guided DNA endonuclease enzymeassociated with the CRISPR (Clustered Regularly Interspersed PalindromicRepeats) adaptive immunity system in Streptococcus pyogenes, among otherbacteria. Cas9 can cleave nearly any sequence complementary to the guideRNA. Includes Cas9 nucleic acid molecules and proteins. Cas9 sequencesare publically available, for example from the GENBANK® sequencedatabase (e.g., GENBANK® Accession Nos. NP_269215.1 and AKS40378.1provide exemplary Cas9 protein sequences, while GENBANK® Accession No.NC_002737.2 provides an exemplary Cas9 nucleic acid sequence therein).One of ordinary skill in the art can identify additional Cas9 nucleicacid and protein sequences, including Cas9 variants.

Differentiation: Refers to the process whereby relatively unspecializedcells (such as embryonic stem cells or other stem cells) acquirespecialized structural and/or functional features characteristic ofmature cells. Similarly, “differentiate” refers to this process.Typically, during differentiation, cellular structure alters andtissue-specific proteins appear.

Embryoid Bodies: Three-dimensional aggregates of pluripotent stem cells.These cells can undergo differentiation into cells of the endoderm,mesoderm and ectoderm. In contrast to monolayer cultures, the spheroidstructures that are formed when pluripotent stem cells aggregate enablesthe non-adherent culture of EBs in suspension, which is useful forbioprocessing approaches. The three-dimensional structure, including theestablishment of complex cell adhesions and paracrine signaling withinthe EB microenvironment, enables differentiation and morphogenesis.

Donor polynucleotide: A polynucleotide that is capable of specificallyinserting into a genomic locus.

Downstream: A relative position on a polynucleotide, wherein the“downstream” position is closer to the 3′ end of the polynucleotide thanthe reference point. In the instance of a double-strandedpolynucleotide, the orientation of 5′ and 3′ ends are based on the sensestrand, as opposed to the antisense strand.

Embryonic stem cells: Embryonic cells derived from the inner cell massof blastocysts or morulae, optionally that have been serially passagedas cell lines. The term includes cells isolated from one or moreblastomeres of an embryo, preferably without destroying the remainder ofthe embryo. The term also includes cells produced by somatic cellnuclear transfer. “Human embryonic stem cells” (hES cells) includesembryonic cells derived from the inner cell mass of human blastocysts ormorulae, optionally that have been serially passaged as cell lines. ThehES cells may be derived from fertilization of an egg cell with sperm orDNA, nuclear transfer, parthenogenesis, or by means to generate hEScells with homozygosity in the HLA region. Human ES cells can beproduced or derived from a zygote, blastomeres, or blastocyst-stagedmammalian embryo produced by the fusion of a sperm and egg cell, nucleartransfer, parthenogenesis, or the reprogramming of chromatin andsubsequent incorporation of the reprogrammed chromatin into a plasmamembrane to produce an embryonic cell. Human embryonic stem cellsinclude, but are not limited to, MAO1, MAO9, ACT-4, No. 3, H1, H7, H9,H14 and ACT30 embryonic stem cells. Human embryonic stem cells,regardless of their source or the particular method used to producethem, can be identified based on (i) the ability to differentiate intocells of all three germ layers, (ii) expression of at least Oct-4 andalkaline phosphatase, and (iii) ability to produce teratomas whentransplanted into immunocompromised animals.

Expand: A process by which the number or amount of cells in a cellculture is increased due to cell division. Similarly, the terms“expansion” or “expanded” refers to this process. The terms“proliferate,” “proliferation” or “proliferated” may be usedinterchangeably with the words “expand,” “expansion”, or “expanded.”Typically, during an expansion phase, the cells do not differentiate toform mature cells, but divide to form more cells.

Expression: The process by which the coded information of a gene isconverted into an operational, non-operational, or structural part of acell, such as the synthesis of a protein. Gene expression can beinfluenced by external signals. For instance, exposure of a cell to ahormone may stimulate expression of a hormone-induced gene. Differenttypes of cells can respond differently to an identical signal.Expression of a gene also can be regulated anywhere in the pathway fromDNA to RNA to protein. Regulation can include controls on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization or degradation of specific protein molecules afterthey are produced.

Feeder layer: Non-proliferating cells (such as irradiated cells) thatcan be used to support proliferation of stem cells. Protocols for theproduction of feeder layers are known in the art, and are available onthe internet, such as at the National Stem Cell Resource website, whichis maintained by the American Type Culture Collection (ATCC).

Growth medium or expansion medium: A synthetic set of culture conditionswith the nutrients necessary to support the growth (cellproliferation/expansion) of a specific population of cells. In oneembodiment, the cells are stem cells, such as iPSCs. Growth mediagenerally include a carbon source, a nitrogen source and a buffer tomaintain pH. In one embodiment, growth medium contains a minimalessential media, such as DMEM, supplemented with various nutrients toenhance stem cell growth. Additionally, the minimal essential media maybe supplemented with additives such as horse, calf or fetal bovineserum.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Isolated: An “isolated” biological component, such as a nucleic acid,protein or organelle that has been substantially separated or purifiedaway from other biological components in the environment (such as acell) in which the component naturally occurs, i.e., chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids andproteins. Similarly, an “isolated” cell has been substantiallyseparated, produced apart from, or purified away from other cells of theorganism in which the cell naturally occurs. Isolated cells can be, forexample, at least 99%, at least 98%, at least 97%, at least 96%, 95%, atleast 94%, at least 93%, at least 92%, or at least 90% pure.

Mammal: This term includes both human and non-human mammals. Examples ofmammals include, but are not limited to: humans and veterinary andlaboratory animals, such as pigs, cows, goats, cats, dogs, rabbits andmice.

Marker or Label: An agent capable of detection, for example by ELISA,spectrophotometry, flow cytometry, immunohistochemistry,immunofluorescence, microscopy, Northern analysis or Southern analysis.For example, a marker can be attached to a nucleic acid molecule orprotein, thereby permitting detection of the nucleic acid molecule orprotein. Examples of markers include, but are not limited to,radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors,ligands, chemiluminescent agents, fluorophores, haptens, enzymes, andcombinations thereof. Methods for labeling and guidance in the choice ofmarkers appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998).

In some embodiments, the marker is a fluorophore (“fluorescent label”).Fluorophores are chemical compounds, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.”

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

Pluripotent stem cells: Stem cells that: (a) are capable of inducingteratomas when transplanted in immunodeficient (SCID) mice; (b) arecapable of differentiating to cell types of all three germ layers (e.g.,can differentiate to ectodermal, mesodermal, and endodermal cell types);and (c) express one or more markers of embryonic stem cells (e.g.,express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc), but thatcannot form an embryo and the extraembryonic membranes (are nottotipotent).

Exemplary pluripotent stem cells include embryonic stem cells derivedfrom the inner cell mass (ICM) of blastocyst stage embryos, as well asembryonic stem cells derived from one or more blastomeres of a cleavagestage or morula stage embryo (optionally without destroying theremainder of the embryo). These embryonic stem cells can be generatedfrom embryonic material produced by fertilization or by asexual means,including somatic cell nuclear transfer (SCNT), parthenogenesis, andandrogenesis. PSCs alone cannot develop into a fetal or adult animalwhen transplanted in utero because they lack the potential to contributeto all extraembryonic tissue (e.g., placenta in vivo or trophoblast invitro).

Pluripotent stem cells also include “induced pluripotent stem cells(iPSCs)” generated by reprogramming a somatic cell by expressing orinducing expression of a combination of factors (herein referred to asreprogramming factors). iPSCs can be generated using fetal, postnatal,newborn, juvenile, or adult somatic cells. In certain embodiments,factors that can be used to reprogram somatic cells to pluripotent stemcells include, for example, Oct4 (sometimes referred to as Oct 3/4),Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somaticcells are reprogrammed by expressing at least two reprogramming factors,at least three reprogramming factors, or four reprogramming factors toreprogram a somatic cell to a pluripotent stem cell. iPSCs are similarin properties to embryonic stem cells.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) ofany length. Therefore, a polynucleotide includes oligonucleotides, andalso gene sequences found in chromosomes. An “oligonucleotide” is aplurality of joined nucleotides joined by native phosphodiester bonds.An oligonucleotide is a polynucleotide of between 6 and 300 nucleotidesin length. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Polypeptide: Three or more covalently attached amino acids. The termencompasses proteins, protein fragments, and protein domains. A“DNA-binding” polypeptide is a polypeptide with the ability tospecifically bind DNA.

The term “polypeptide” is specifically intended to cover naturallyoccurring proteins, as well as those which are recombinantly orsynthetically produced. The term “functional fragments of a polypeptide”refers to all fragments of a polypeptide that retain an activity of thepolypeptide. Biologically functional fragments, for example, can vary insize from a polypeptide fragment as small as an epitope capable ofbinding an antibody molecule to a large polypeptide capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An “epitope” is a region of apolypeptide capable of binding an immunoglobulin generated in responseto contact with an antigen. Thus, smaller peptides containing thebiological activity of insulin, or conservative variants of the insulin,are thus included as being of use.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Examples ofconservative substitutions are shown below.

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, should be minimized in order to preservethe functional and immunologic identity of the encoded protein. Theimmunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody; a variant that is recognized bysuch an antibody is immunologically conserved. Any cDNA sequence variantwill preferably introduce no more than twenty, and preferably fewer thanten amino acid substitutions into the encoded polypeptide. Variant aminoacid sequences may, for example, be 80%, 90% or even 95% or 98%identical to the native amino acid sequence.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription.

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), an inducible promoter(i.e., a promoter whose state, active/“ON” or inactive/“OFF”, iscontrolled by an external stimulus, e.g., the presence of a particulartemperature, compound, or protein.), a spatially restricted promoter(e.g., tissue specific promoter, cell type specific promoter, etc.), orit may be a temporally restricted promoter (i.e., the promoter is in the“ON” state or “OFF” state during specific stages of embryonicdevelopment or during specific stages of a biological process, e.g.,hair follicle cycle in mice).

Examples of inducible promoters include, but are not limited to T7 RNApolymerase promoter, T3 RNA polymerase promoter,isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, tetracycline-regulatedpromoter, steroid-regulated promoters, metal-regulated promoters,estrogen receptor-regulated promoter, etc. Inducible promoters can beregulated by molecules including, but not limited to, doxycycline; RNApolymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogenreceptor fusion; etc.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell. For example, apreparation of a protein is purified such that the protein represents atleast 50% of the total protein content of the preparation. Similarly, apurified oligonucleotide preparation is one in which the oligonucleotideis more pure than in an environment including a complex mixture ofoligonucleotides. A purified population of nucleic acids or proteins isgreater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% pure, or free other nucleic acids or proteins, respectively.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. Similarly, arecombinant protein is one coded for by a recombinant nucleic acidmolecule.

Recombination: A process of exchange of genetic information between twopolynucleotides. “Homologous recombination (HR)” refers to thespecialized form of an exchange that takes place, for example, duringrepair of double-strand breaks in cells. Nucleotide sequence homology isutilized in recombination, for example using a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target.“Recombination efficiency” is the rate and effectiveness ofrecombination a particular host cells, such as an iPSC.

Enzyme mismatch cleavage assays can be used to quantify the efficiencyof mutations induced by Cas9, namely, T7E1 and Surveyor. This test showsthe percent of insertion or deletion of bases in the DNA (Indels) (seeZhou et al., Nature. 2014 May 22; 509(7501):487-91, incorporated hereinby reference). In this system, 9-50% of indels efficiency is consideredas high rate efficiency recombination.

A widely used method to identify mutations is the T7 Endonuclease I(T7E1) mutation detection assay. This assay detects heteroduplex DNAthat results from the annealing of a DNA strand, including desiredmutations, with a wildtype DNA strand. In some embodiments, this assayis used to quantify the efficiency of mutations induced by Cas9. (seeZhou et al., Nature. 2014 May 22; 509(7501):487-91, incorporated hereinby reference).

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a FGF polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul, et al., Nature Genet., 6:119, 1994 presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul, et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%,sequence identity counted over the full length alignment with the aminoacid sequence of the factor using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Short Guide RNA (gRNA): Short guide RNA used in conjunction with CRISPRassociated systems (Cas). sgRNAs contains nucleotides of sequencecomplementary to the desired target site. Watson-crick pairing of thesgRNA with the target site recruits the nuclease-deficient Cas9 to bindthe DNA at that locus.

Subject: Human and non-human animals, including all vertebrates, such asmammals and non-mammals, such as non-human primates, mice, rabbits,sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. Inmany embodiments of the described methods, the subject is a human.

Transgene: An exogenous gene.

Treating, Treatment, and Therapy: Any success or indicia of success inthe attenuation or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement,remission, diminishing of symptoms or making the condition moretolerable to the patient, slowing in the rate of degeneration ordecline, making the final point of degeneration less debilitating,improving a subject's physical or mental well-being, or improvingvision. The treatment may be assessed by objective or subjectiveparameters; including the results of a physical examination,neurological examination, or psychiatric evaluations.

Undifferentiated: Cells that display characteristic markers andmorphological characteristics of undifferentiated cells, distinguishingthem from differentiated cells of embryo or adult origin. Thus, in someembodiments, undifferentiated cells do not express cell lineage specificmarkers.

Upstream: A relative position on a polynucleotide, wherein the“upstream” position is closer to the 5′ end of the polynucleotide thanthe reference point. In the instance of a double-strandedpolynucleotide, the orientation of 5′ and 3′ ends are based on the sensestrand, as opposed to the antisense strand.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

“Lentiviral vector” refers to a gene delivery vehicle adapted fromlentiviruses, a subclass of Retroviruses. Lentiviruses have recentlybeen adapted as gene delivery vehicles (vectors) thanks to their abilityto integrate into the genome of non-dividing cells, which is the uniquefeature of lentiviruses as other retroviruses can infect only dividingcells. The viral genome in the form of RNA is reverse-transcribed whenthe virus enters the cell to produce DNA, which is then inserted intothe genome at a random position by the viral integrase enzyme. Thevector, now called a provirus, remains in the genome and is passed on tothe progeny of the cell when it divides. Generally, lentiviral vectorsdo not include the genes required for their replication, and thus are“replication defective.” To produce a lentivirus, several plasmids aretransfected into a so-called packaging cell line, for example HEK 293.One or more plasmids, generally referred to as packaging plasmids,encode the virion proteins, such as the capsid and the reversetranscriptase. Another plasmid contains the genetic material to bedelivered by the vector. It is transcribed to produce thesingle-stranded RNA viral genome and is marked by the presence of the φ(psi) sequence. This sequence is used to package the genome into thevirion.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle.Viral vectors are known in the art, and include, for example,adenovirus, AAV, lentivirus and herpes virus.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

Methods for Producing Induced Pluripotent Stem Cells (iPSC)

iPSC cells can be indefinitely maintained in vitro in anundifferentiated state and yet are capable of differentiating intovirtually any cell type. Methods are provided herein wherein somaticcells are used to prepare induced pluripotent stem cells that are highlyefficient for knock-in and/or knock out of one or more genes ofinterest. Disclosed herein are methods to induce the production of theseiPSC, such as human iPSC.

Somatic Cells

The starting somatic cell can be any cell of interest. Any cells otherthan germ cells of mammalian origin (such as, humans, mice, monkeys,pigs, rats etc.) can be used as starting material for the production ofiPSCs. In one embodiment, the stem cells are human Examples includekeratinizing epithelial cells, mucosal epithelial cells, exocrine glandepithelial cells, endocrine cells, liver cells, epithelial cells,endothelial cells, fibroblasts, muscle cells, cells of the blood and theimmune system, cells of the nervous system including nerve cells andglia cells, pigment cells, and progenitor cells, including hematopoieticstem cells, amongst others. There is no limitation on the degree of celldifferentiation, the age of an animal from which cells are collected andthe like; even undifferentiated progenitor cells (including somatic stemcells) and finally differentiated mature cells can be used alike assources of somatic cells in the present invention. The somatic cell canbe an adult or a fetal cell. In a specific non-limiting example, thesomatic cell is a fibroblast. In another specific non-limiting example,the somatic cell is a hepatocyte.

The choice of mammalian individuals as a source of somatic cells is notparticularly limited. Allogenic cells can be used, if the resultingcells will be transplanted into a subject. Thus, in some embodiments,the iPSCs are not matched for MHC (e.g., HLA) to a subject. In someembodiments, when the iPSCs obtained are to be used for regenerativemedicine in humans, cells can be collected from the somatic cells fromthe subject to be treated, or another subject with the same orsubstantially the same HLA type as that of the patient. Thus, the stemcells can be autologous or substantially the same HLA type.“Substantially the same HLA type” indicates that the HLA type of donormatches with that of a patient to the extent that the transplantedcells, which have been obtained by inducing differentiation of iPSCsderived from the donor's somatic cells, can be engrafted when they aretransplanted to the subject. The subject optionally can be treated withan immunosuppressant. In one example, it includes an HLA type whereinmajor HLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR, thefour major loci further including HLA-Cw) are identical.

Somatic cells isolated from a mammal can be pre-cultured using a mediumknown to be suitable for their cultivation according to the choice ofcells before being subjected to the step of nuclear reprogrammingSpecific non-limiting examples of such media include, but are notlimited to, minimal essential medium (MEM) containing about 5 to 20%fetal calf serum (FCS), Dulbecco's modified Eagle medium (DMEM),RPMI1640 medium, 199 medium, F12 medium, and the like. One of skill inthe art can readily ascertain appropriate tissue culture conditions topropagate particular cell types from a mammal, such as a human. In someembodiments, to obtain completely xeno-free human iPSCs, the medium canexclude ingredients derived from non-human animals, such as FCS. Mediacomprising a basal medium supplemented with human-derived ingredientssuitable for cultivation of various somatic cells (particularly,recombinant human proteins such as growth factors), non-essential aminoacids, vitamins and the like are commercially available; those skilledin the art are able to choose an appropriate xeno-free medium accordingto the source of somatic cells. Somatic cells pre-cultured using axeno-free medium are dissociated from the culture vessel using anappropriate xeno-free cell dissociation solution, and recovered, afterwhich they are brought into contact with nuclear reprogrammingsubstances.

Generally, cells are cultured at about 35 to 38° C., usually at 37° C.,in about 4-6% CO₂, generally at 5% CO₂, unless specifically indicatedotherwise below.

Constructs Including a Doxycycline Inducible Promoter Operably Linked toa Nucleic Acid Molecule Encoding Cas9

In some embodiments, the somatic cells is transfected to introduce anucleic acid molecule including a doxycycline promoter operably linkedto a nucleic acid encoding Cas9. One skilled in the art will recognizethat any Cas9 protein can be used in the systems and methods disclosedherein. This promoter provides for inducible expression of Cas9. In aTet-On system, the rtTA protein is capable of binding the operator (thedoxycycline promoter) only if bound by a tetracycline. Thus, thepromoter is activated by doxycycline. The systems disclosed hereinutilize an inducible expression platform based on 3G TET technology. Anexemplary nucleic acid sequence of this promoter is shown below (SEQ IDNO: 1).

(SEQ ID NO: 1) ATCGATACTAGACTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAVariants of this nucleic acid sequence can also be used, such as nucleicacid sequences at least 90%, 91%, 92%, 935, 94%, 95%, 96%, 97%, 98% or99% sequence identical to SEQ ID NO: 1, provided the nucleic acidsequence functions as a doxycycline inducible promoter.

A doxycycline inducible promoter is a highly sensitive and providestranscription without leakiness. Inducible genetic engineering can beused, using the method disclosed herein, to produce a knockdown, knockinor dual knockins-knockdowns in genes of interest. One form of adoxycycline inducible promoter is the Tet-on-3G system; this system isof use in the methods disclosed herein. This system is composed of thesetwo elements: (1) a reverse tetracycline-controlled transactivatorinducible promoter (rtTA) expressed constitutively, under the control ofa promoter, such as a Ubiquitin C promoter; (2) a Tetracycline ResponseElement (TRE) controlling the transcription of a sequence of interest.In some embodiments, the TRE is composed of 7 repeats of the 19 bpbacterial tet-O sequence placed upstream of a minimal promoter with verylow basal expression in the absence of Tet-On. The rtTA protein bindsthe TRE only if bound by a doxycycline. The addition of doxycycline tothe system initiates the transcription of the sequence of interest(fluorescent reporter genes; Cas9 etc.). An exemplary construct is shownin FIG. 1. Additional suitable promoters are disclosed, for example, inPublished U.S. Patent Application No. 2014/0107190, which isincorporated herein by reference. Thus, in some embodiments, the somaticcell includes a construct encoding the rtTA protein, and a TREcontrolling the transcription of Cas9. Tetracycline/doxycline induciblepromoters are disclosed, for example, in U.S. Pat. Nos. 5,464,758;5,851,796; 5,912,411; and 6,000,494, all incorporated by referenceherein. Any of these promoters are of use in the methods disclosedherein.

In some embodiments, a doxycycline inducible promoter operably linked toCas9 is introduced into the somatic cell. One Cas9 of use is fromStreptococcus pyogenes as depicted in SEQ ID NO. 2 below.

(SEQ ID NO: 2) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD.Variants of this amino acid sequence can also be used, such as aminoacid sequences at least 90%, 91%, 92%, 935, 94%, 95%, 96%, 97%, 98% or99% sequence identical to SEQ ID NO: 2, provided the nucleic acidsequence functions as a Cas9 polypeptide. In some embodiments, thevariant includes at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservativeamino acid substitutions in SEQ ID NO: 2.

In other embodiments, the Streptococcus pyogenes Cas9 peptide caninclude one or more of the mutations described in the literature,including but not limited to the functional mutations described in:Fonfara et al. Nucleic Acids Res. 2014 February; 42(4):2577-90;Nishimasu H. et al. Cell. 2014 Feb. 27; 156(5):935-49; Jinek M et al.Science. 2012 Aug. 17; 337(6096):816-21; and Jinek M. et al. Science.2014 Mar. 14; 343(6176). Thus in some embodiments the systems andmethods disclosed herein can be used with the wild type Cas9 proteinhaving double-stranded nuclease activity, Cas9 mutants that act assingle stranded nickases, or other mutants with modified nucleaseactivity.

The Cas9 peptide can be an activating Cas9 (Cas9a). Suitable Cas9sequences include SpCas9-HF1, dCas9-VP64. Suitable Cas9 molecules aredisclosed, for example, in Chavez et al., Nat. Methods 12: 326-328, Oct.1, 2015, which is incorporated herein by reference. Optionally, asynergistic activator can be encoded with the Cas9, see the internet,sam.genome-engineering.org, incorporated herein by reference.

CRISPR-Cas9 uses a short guide RNA (sgRNA) to direct nuclease Cas9 tothe target site and generate double-strand breaks, stimulating DNArepair processes that give rise to DNA editing. To circumvent offtargets effects, a modified Cas9 can be utilized, without any reportedoff target effect (SpCas9-HF1). SpCas9-HF1 enables loss, but also gainof function, provided that the desired template sequence is deliveredand used by the Homology Directed Repair cell machinery. Additionally,SpCas9-HF1 can be used for whole genome loss-of-function screening usingsgRNA libraries. To enable gain-of-function for whole genome screening,a CRISPR-Cas9 Synergistic Activation Mediator (SAM) complex can be used.This is a protein complex composed of an inactive Cas9-VP64 fusion andactivation helper proteins (MS2-P65-HSF1). This complex interacts withsgRNA to ensure robust transcriptional activation of target genes. Thissystem can be used in the present methods for gain-of-functionscreening.

Cas9 can be used for inhibiting genes (Cas9i). This is a catalyticallyactive Cas9 that, when guided with sgRNA, will induce loss of functionby site-specifically cleavage of double-stranded DNA, resulting in theactivation of the doublestrand break (DSB) repair machinery. Thus, useof Cas9 results in loss of gene function. A single or a library of gRNAcan be used for loss-of-function screens. CRISPR knockout libraries orsingle gRNA render genes non-functional by inducing insertions ordeletions in targeted genes.

The Cas9 includes a catalytically active nuclease domain. In someembodiments, the Cas9 nuclease includes an HNH-like endonuclease and aRuvC-like endonuclease. Thus in some embodiments, to generate adouble-stranded DNA break, the HNH-like endonuclease cleaves the DNAstrand complementary to the sgRNA, and the RuvC-like domain cleaves thenon-complementary DNA strand. A Cas9 endonuclease can be guided tospecific genomic targets using specific sgRNA (see below).

Optionally, a nucleic acid molecule encoding a marker also can beoperably linked to the doxycycline inducible promoter, or to anotherpromoter. Markers include, but are not limited to, enzymes andfluorescent proteins. A marker may be a protein (including secreted,cell surface, or internal proteins; either synthesized or taken up bythe cell); a nucleic acid (such as an mRNA, or enzymatically activenucleic acid molecule) or a polysaccharide. Included are determinants ofany such cell components that are detectable by antibody, lectin, probeor nucleic acid amplification reaction that are specific for the markerof the cell type of interest. The markers can also be identified by abiochemical or enzyme assay or biological response that depends on thefunction of the gene product. Nucleic acid sequences encoding thesemarkers can be operably linked to the promoter. In addition, other genescan be included, such as genes that may influence stem cell todifferentiate, or influence function, or physiology.

In specific non-limiting examples, the marker is tdTomato fluorescentprotein or green fluorescent protein. In other embodiments, a nucleicacid molecule encoding a marker is not operably linked the doxycyclinepromoter.

In some embodiments, the doxycycline promoter operably linked to thenucleic acid encoding Cas9 are included in a vector. Plasmids have beendesigned with a number of goals in mind, such as achieving regulatedhigh copy number and avoiding potential causes of plasmid instability inbacteria, and providing means for plasmid selection that are compatiblewith use in mammalian cells, including human cells. Particular attentionhas been paid to the dual requirements of plasmids for use in humancells. First, they are suitable for maintenance and fermentation in E.coli, so that large amounts of DNA can be produced and purified. Second,they are safe and suitable for use in human patients and animals. Thefirst requirement calls for high copy number plasmids that can beselected for and stably maintained relatively easily during bacterialfermentation. The second requirement calls for attention to elementssuch as selectable markers and other coding sequences. In someembodiments plasmids of use are composed of: (1) a high copy numberreplication origin, (2) a selectable marker, such as, but not limitedto, the neo gene for antibiotic selection, such as with kanamycin,puromycin, neomycin, (3) transcription termination sequences, includingthe tyrosinase enhancer and (4) a multicloning site for incorporation ofvarious nucleic acid cassettes; and (5) a nucleic acid sequence encodinga marker operably linked to the tyrosinase promoter. There are numerousplasmid vectors that are known in the art for inducing a nucleic acidencoding a protein. These include, but are not limited to, the vectorsdisclosed in U.S. Pat. Nos. 6,103,470; 7,598,364; 7,989,425; and6,416,998, which are incorporated herein by reference.

Viral vectors can be utilized for the introduction of nucleic acids,including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377), human herpesvirus vectors(HHV) such as HHV-6 and HHV-7, and retroviruses of avian (Brandyopadhyayet al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J.Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437;Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J.Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors can be used. Vectors can be obtained from commercialsources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp.,Meriden, Conn.; Stratagene, La Jolla, Calif.). Suitable vectors aredisclosed, for example, in U.S. Published Patent Application No.2010/0247486, which is incorporated herein by reference. In specificnon-limiting examples, the vectors are retrovirus vectors (for example,lentivirus vectors), measles virus vectors, alphavirus vectors,baculovirus vectors, Sindbis virus vectors, adenovirus and poliovirusvectors.

In some embodiments, the vector is a lentiviral vector. An advantage oflentiviruses for infection of cells is the ability for sustainedtransgene expression. Leintiviruses include, but are not limited to,Human Immunodeficiency Virus type 1 (HIV-1), Human ImmunodeficiencyVirus type 2 (HIV-2), Simian Immunodeficiency Virus (SIV), FelineImmunodeficiency Virus (FIV), Equine Infectious Anaemia Virus (EIAV),Bovine Immunodeficiency Virus (BIV), Visna Virus of sheep (VISNA) andCaprine Arthritis-Encephalitis Virus (CAEV). Lentiviral vectors are wellknown in the art (see, for example, Naldini et al., Science,272(5259):263-267, 1996; Zufferey et al., Nat Biotechnol, 15(9):871-875,1997; Blomer et al., J Virol, 71(9):6641-6649, 1997; U.S. Pat. Nos.6,013,516 and 5,994,136). Recombinant lentiviral vectors are capable ofinfecting non-dividing cells and can be used for both in vivo and invitro gene transfer and expression of nucleic acid sequences. Forexample, recombinant lentivirus capable of infecting a non-dividing cellwherein a suitable host cell is transfected with two or more vectorscarrying the packaging functions, namely gag, pol and env, as well asrev and tat is described in U.S. Pat. No. 5,994,136, incorporated hereinby reference.

A recombinant lentivirus can be targeted to a specific cell type bylinkage of the envelope protein with an antibody or a particular ligandfor targeting to a receptor of a particular cell-type. A sequence(including a regulatory region) of interest is inserted into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, in order to produce a target-specific vector.The recombinant lentiviruses can be genetically modified in such a waythat certain genes constituting the native infectious virus areeliminated and replaced with a nucleic acid sequence of interest to beintroduced into the target cells.

In some embodiments, a lentiviral vector can integrate into the genomeof the host cell. The genetic material thus transferred is thentranscribed and possibly translated into proteins inside the host cell.In other embodiments, a lentiviral vector is a non integrativelentiviral vector, such that the vector is present in episomal forms.

The lentiviral vector can further comprise additional elements whichhelp to improve expression of the genes encoded within the vector.Regions required for the integration of the vector into the genome ofthe target cell such as the Long-terminal repeats (LTRs). Thus, alentiviral vector can include a 5′ LTR and a 3′ LTR. “5′ LTR” refers toa 5′ retroviral or lentiviral long terminal repeat, which may or may notbe modified from its corresponding native 5′ LTR by deleting and/ormutating endogenous sequences and/or adding heterologous sequences. The5′ LTR may be natural or synthetic. “3′ LTR” refers to a 3′ retroviralor lentiviral long terminal repeat, which may or may not be modifiedfrom its corresponding native (i.e., that existing in the wild-typeretrovirus) 3′ LTR by deleting and/or mutating endogenous sequencesand/or adding heterologous sequences. The 3′ LTR may be natural orsynthetic.

An encapsidation sequence such as the lentiviral Psi (ψ) sequence can beincluded in the vector. In some embodiments, sequences enhancing the RNAnuclear export, such as the sequence comprising the HIV-1 REV responseelement (RRE) sequence, can be included in the vector. Another sequencethat enhances the RNA nuclear export is the CTE sequence (Oh et al,2007, Retrovirology. 2007 Jun. 5; 4:38.). These sequences are alsouseful for determining the copy number of the integrated lentiviralvectors. Other sequences that enhance DNA nuclear import are lentiviralcPPT CTS sequences from HIV-2, SIV, FIV, EIAV, BIV, VISNA and CAEV. Anyof these sequences can be included in the vector.

In another embodiment the lentiviral vector is another form ofself-inactivating (SIN) vector as a result of a deletion in the 3′ longterminal repeat region (LTR). In some examples, the vector contains adeletion within the viral promoter. The LTR of lentiviruses such as theHIV LTR contains a viral promoter. Although this promoter is relativelyinefficient, when transactivated by e.g. tat, the promoter is efficientbecause tat-mediated transactivation increases the rate of transcriptionabout 100 fold. In some circumstances, the presence of the viralpromoter can interfere with transcription of heterologous promotersoperably linked to a transgene. To minimize such interference and betterregulate the expression of transgenes, the lentiviral promoter may bedeleted.

In some embodiments, the lentiviral vector comprises, in the 5′ to 3′orientation: the 5′ LTR (wild-type or modified), A Rev response element(RRE), a c polypurine tract (cPPT), the transcriptional regulatoryregion, the doxycycline promoter linked to Cas9, an optionaltranscriptional regulation element, and the 3′ LTR.

Methods of transfection of DNA include calcium phosphate coprecipitates,conventional mechanical procedures such as microinjection,electroporation, insertion of a plasmid encased in liposomes, or virusvectors.

A viral gene delivery system can be an RNA-based or DNA-based viralvector. An episomal gene delivery system can be a plasmid, anEpstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, anadenovirus-based vector, a simian virus 40 (SV40)-based episomal vector,a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.

Markers include, but are not limited to, fluorescence proteins (forexample, green fluorescent protein or red fluorescent protein), enzymes(for example, horse radish peroxidase or alkaline phosphatase orfirefly/renilla luciferase or nanoluc), or other proteins.

Reprogramming

Somatic cells can be reprogrammed to produce induced pluripotent stemcells (iPSCs) using methods known to one of skill in the art. One ofskill in the art can readily produce induced pluripotent stem cells, seefor example, Published U.S. Patent Application No. 20090246875,Published U.S. Patent Application No. 2010/0210014; Published U.S.Patent Application No. 20120276636; U.S. Pat. Nos. 8,058,065; 8,129,187;8,278,620; PCT Publication NO. WO 2007/069666 A1, and U.S. Pat. No.8,268,620, all of which are incorporated herein by reference. Generally,nuclear reprogramming factors are used to produce pluripotent stem cellsfrom a somatic cell. In some embodiments, at least three, or at leastfour, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized. Inother embodiments, Oct3/4, Sox2, c-Myc and Klf4 is utilized.

The cells are treated with a nuclear reprogramming substance, which isgenerally one or more factor(s) capable of inducing an iPSC from asomatic cell or a nucleic acid that encodes these substances (includingforms integrated in a vector). The nuclear reprogramming substancesgenerally include at least Oct3/4, Klf4 and Sox2 or nucleic acids thatencode these molecules. A functional inhibitor of p53, L-myc or anucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acidthat encodes Lin28 or Lin28b, can be utilized as additional nuclearreprogramming substances. Nanog can also be utilized for nuclearreprogramming. As disclosed in published U.S. Patent Application No.2012/0196360, exemplary reprogramming factors for the production ofiPSCs include (1) Oct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced withSox1, Sox3, Sox15, Sox17 or Sox18; Klf4 is replaceable with Klf1, Klf2or Klf5); (2) Oct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen(SV40LT); (3) Oct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus(HPV)16 E6; (4) Oct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3/4,Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3/4, Klf4, Sox2,L-Myc, TERT, Bmi1; (7) Oct3/4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3/4,Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3/4, Klf4, Sox2, L-Myc, Lin28,TERT, SV40LT; (10) Oct3/4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3/4,Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3/4, Klf4,Sox2; (13) Oct3/4, Klf4, Sox2, TERT, SV40LT; (14) Oct3/4, Klf4, Sox2,TERT, HPV16 E6; (15) Oct3/4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3/4,Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3/4, Klf4, Sox2, TERT,Bmi1; (18) Oct3/4, Klf4, Sox2, Lin28 (19) Oct3/4, Klf4, Sox2, Lin28,SV40LT; (20) Oct3/4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3/4, Klf4,Sox2, SV40LT; or (22) Oct3/4, Esrrb, Sox2 (Esrrb is replaceable withEsrrg). In one non-limiting example, Oct3/4, Klf4, Sox2, and c-Myc areutilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized, seefor example, U.S. Pat. No. 7,682,828, which is incorporated herein byreference. These factors include, but are not limited to, Oct3/4, Klf4and Sox2. In other examples, the factors include, but are not limited toOct 3/4, Klf4 and Myc. In some non-limiting examples, Oct3/4, Klf4,c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3/4,Klf4, Sox2 and Sal 4 are utilized.

Mouse and human cDNA sequences of these nuclear reprogramming substancesare available with reference to the NCBI accession numbers mentioned inWO 2007/069666, which is incorporated herein by reference. Methods forintroducing one or more reprogramming substances, or nucleic acidsencoding these reprogramming substances, are known in the art, anddisclosed for example, in published U.S. Patent Application No.2012/0196360 and U.S. Pat. No. 8,071,369, which both are incorporatedherein by reference.

After being cultured with nuclear reprogramming substances, the cellcan, for example, be cultured under conditions suitable for culturingstem cells. In the case of mouse cells, the culture is carried out withthe addition of Leukemia Inhibitory Factor (LIF) as a differentiationsuppression factor to an ordinary medium. In the case of human cells, itis desirable that basic fibroblast growth factor (bFGF) be added inplace of LIF.

In some embodiments, the cell is cultured in the co-presence of mouseembryonic fibroblasts treated with radiation or an antibiotic toterminate the cell division, as feeder cells. Mouse embryonicfibroblasts in common use as feeders include the STO cell line (ATCCCRL-1503) and the like; for induction of an iPSC, useful cells can begenerated by stably integrating the neomycin resistance gene and the LIFgene in the STO cell (SNL76/7 STO cell; ECACC 07032801) (McMahon, A. P.& Bradley, A. Cell 62, 1073-1085, 1990) and the like can be used.Mitomycin C-treated MEFs are commercially available from Millipore.Gamma-irradiated MEFs are commercially available from Global StemGenerally, somatic cells are transduced with reprogramming factors inthe absence of MEFs. In some embodiments, about 7 to eight days aftertransduction, the cells are re-seeded onto MEFs.

The expression of a key pluripotency factor, NANOG, and embryonic stemcell specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) havebeen routinely used to identify fully reprogrammed human cells. At thefunctional level, iPSCs also demonstrate the ability to differentiateinto lineages from all three embryonic germ layers.

In some embodiments, upon inducing the somatic cells to produce thehuman iPSC, more than 10% of the human induced pluripotent stem cellsexpress the Cas9 when the cells are exposed to doxycycline. Inadditional embodiments, more than about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, or about 50% of the human inducedpluripotent stem cells express the Cas9 when the cells are exposed todoxycycline. In specific non-limiting examples, about 35% to about 45%of the human induced pluripotent stem cells express the Cas9 when thecells are exposed to doxycycline, such as about 38% to about 42%, suchas about 40%. In this context, “about” indicates within one percent. Inother embodiments, more than 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% ofthe human induced pluripotent stem cell clones or colonies express theCas9 when the cells are exposed to doxycycline. In specific non-limitingexamples, 35% to 45% of the human induced pluripotent stem cell clonesor colonies express the Cas9 when the cells are exposed to doxycycline,such as 38% to 42%, such as 40%.

Differentiation of iPSC

The iPSC can be differentiated into any cell type of interest.Appropriate differentiated cells (of ectodermal, mesodermal orendodermal lineage) can be produced. These cells are of use in modelingsimple and complex diseases and for treatment in a variety of forms. Fortreatment, the mode of administration can be determined by a person ofskill in the art depending on the type of organ/injury to be treated.For example, iPSCs or differentiated cells derived therefrom, may beadministered by injection (as a suspension) or implanted on abiodegradable matrix.

In some embodiments, iPSCs can be differentiated into neurons, such asadrenergic or dopaminergic neurons. The iPS cells thus established canbe used for various purposes. For example, by utilizing a method ofdifferentiation iPSC can be differentiated into pancreatic stem-likecells, hematopoietic cells, myocardial cells, myofibroblasts, bloodcells, vascular endothelial cells, insulin-secreting cells and livercells, see for example, U.S. Published Patent Application No.2015/0252330, incorporated herein by reference. Additional methods aredisclosed, for example, in U.S. Published Patent Application No.2016/0083715, U.S. Published Patent Application No. 2015/0368713, U.S.Published Patent Application No. 2015/0159133, U.S. Published PatentApplication No. 2014/0356951, U.S. Published Patent Application No.2013/0295064, which are incorporated herein by reference. In onenon-limiting example, the iPSC are differentiated into hepatocytes.

CRISPR Cas9 Recombination

In some embodiments, the methods also include introducing nucleic acidsencoding guide RNAs (gRNAs). In some embodiments, the methods disclosedherein can include introducing the nucleic acid encoding the sgRNAs intothe somatic cell, prior to inducing formation of an iPSC. In otherembodiments, the methods disclosed herein can include introducing thenucleic acid encoding the sgRNAs into an iPSC including the doxycyclinepromoter operably linked to Cas9. In further embodiments, the methodsdisclosed herein can include introducing the nucleic acid encoding thesgRNAs into a differentiated cell, after inducing the iPSC (includingthe doxycycline promoter operably linked to Cas9) to differentiate.

The nucleic acid encoding the sgRNA can be linked to a constitutivepromoter. Suitable promoters include, but are not limited to, the U6promoter or the ubiquitin promoter.

(SEQ ID NO: 6) CGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACGGTTGTAAATGAGCACACAAAATACACATGCTAAAATATTATATTCTATGACCTTTATAAAATCAACCAAAATCTTCTTTTTAATAACTTTAGTATCAATAATTAGAATTTTTATGTTCCTTTTTGCAAACTTTTAATAAAAATGAGCAAAATAAAAAAACGCTAGTTTTAGTAACTCGCGTTGTTTTCTTCACCTTTAATAATAGCTACTCCACCACTTGTTCCTAAGCGGTCAGCTCCTGCTTCAATCATTTTTTGAGCATCTTCAAATGTTCTAACTCCACCAGCTGCTTTAACTAAAGCATTGTCTTTAACAACTGACTTCATTAGTTTAACATCTTCAAATGTTGCACCTGATTTTGAAAATCCTGTTGATGTTTTAACAAATTCTAATCCAGCTTCAACAGCTATTTCACAAGCTTTCATGATTTCTTCTTTTGTTAATAAACAATTTTCCATAATACATTTAACAACATGTGATCCAGCTGCTTTTTTTACAGCTTTCATGTCTTCTAAAACTAATTCATAATTTTTGTCTTTTAATGCACCAATATTTAATACCATATCAATTTCTGTTGCACCATCTTTAATTGCTTCAGAAACTTCGAATGCTTTTGTAGCTGTTGTGCATGCACCTAGAGGAAAACCTACAACATTTGTTATTCCTACATTTGTGCCTTTTAATAATTCTTTACAATAGCTTGTTCAATATGAATTAACACAAACTGTTGCAA AATCAAATTCAATTGC

Variants of this nucleic acid sequence can also be used, such as nucleicacid sequences at least 90%, 91%, 92%, 935, 94%, 95%, 96%, 97%, 98% or99% sequence identical to SEQ ID NO: 2, provided the nucleic acidsequence functions as a promoter. In some embodiments, primers are usedwhen sequencing nucleic acids encoding sgRNAs into an iPSC or into acell differentiated from the iPSC. These primers include, but are notlimited to:

hU6-F: (SEQ ID NO: 9) 5′-GAGGGCCTATTTCCCATGATT-3′ LKO.1 5′:(SEQ ID NO: 10) 5′-GACTATCATATGCTTACCGT-3′

In other embodiments, an inducible promoter is utilized, and the sgRNAsare introduced into the starting somatic cell. The sgRNA can also beintroduced into cells differentiated from the iPSC. When recombinationis desired, expression can, in some circumstances, be induced from thisinducible promoter. Thus, expression can be induced in the startingsomatic cells, iPSCs, or cells differentiated from the iPSCs. Thesepromoters include, but are not limited to:

Target tissue Promoter Vector Transgene References LIVER Apo A-I Ad ApoA-I [De Geest et al., 2000] ApoE HCAd ApoE [Kim et al., 2001]α₁-antitrypsin (hAAT) Ad Apo A-I [Van Linthout et al., 2002] HCAd hAAT[Schiedner et al., 1998][Scheidner et al., 2002] Plasmid factorIX [Miaoet al., 2001][Eluhardt et al., 2002] hAAT & Apo A-I Retroviral hAAT[Okuyama, 1996] Transthyretin HCAd hGH [Burcin et al., 1999]Liver-enriched Transgenic LUC [Kistner et al., 1996] activator AlbuminHCAd FactorVIII [Reddy et al., 2002] Lentivirus factorIX [Follenzi etal., 2002] Phosphoenolpyruvate HCAd VLDLR [Oka et al., 2001]carboxykinase (PEPCK) RNAP_(n) promoter Retrovirus hAAT [Rettinger etal., 1994] ENDOTHELIUM PAI-1 AAV Thrombomodulin [Mimar J, 2001] ICAM-2,Endoglin Plasmid Endoglin [Velasco et al., 2001] ICAM-2, fit-1, vWF AdlacZ [Nicklin et al., 2001] MUSCLE MCK Ad LacZ, LUC [Hauser et al.,2001][Larochelle et al., 2002] Plasmid hBSAg [Weeratns et al., 2001]Ad/AAV γ-surcoglycan [Cordier et al., 2000] SMC α-actin Plasmid LUC[Keogh et al., 1999][Prentice et al., 1997] Ad Rb/E2F hybrid [Wills etal., 2001] Ad GFP, lacZ, IFNγ [Ribault et al., 2001] AAV Factor IX[Hagstrom et al., 2000] Myosin heavy-chain Plasmid CAT [Skarli et al.,1998] AAV lacZ, hGH [Aikawa et al., 2002] Myosin light-chain Ad LacZ,LUC [Griscelli et al., 1998][Franz et al., 1997] AAV GFP, antisense[Phillips et al., 2002] EPITHELIUM Cytokeratin 18 Plasmid LacZ, CFTR[Chow et al., 1997][Kochler et al., 2001] CFTR Ad LacZ, LUC [Imler etal., 1996][Suzuki et al., 1996] NEURONAL GFAP, NSE, Synapsin Ad LacZ,GFP [Smith-Arica et al., 2000][Glover et al., 2002] I, Preproenkephalin,AAV LUC, GFP [Xu et al., 2001] Dopamine β- Plasmid, Ad CAT, GFP, lacZ[Hwang et al., 2001] hydroxylase (dβH) Prolactin Ad LacZ, HSV-tk[Southgate et al., 2000] Myelin basic protein AAV GFP [Chen et al.,1998] ERYTHROID Ankyrin Retrovirus γ-globin [Sabatino et al., 2001]Lentivirus ferrochelatase [Richard et al., 2001] α-spectrin, GlobinLentivirus GFP, β/γ-globin [Moreau-Gaudry et al., 2001] HLA-DRαLentivirus GFP [Cui et al., 2002] CD4 Retroviral GFP [Zhao-Emonet JC,2000] Dectin-2 Plasmid GFP, LUC [Morita et al., 2001] ABBREVIATIONS:PAI-1, plasminogen activator inhibitor I; ICAM-2, Intercellular adhesionmolecule2; fit-1, fms-like tyrosine kinase-J; vWF, von-Willebrandfactor; MCK, muscle creatine kinase; CFTR, cystic fibrosis transmembraneconductance regulator; GFAP, glial fibrillary acidic protein; NSE,neumoral-specific endolase; LUC, luciferase; GFP, green flourescensprotein; HSV-tk, harpes simplex virus thymidine kinase.Table from Papadkis et al., Current Gene Therapy 4: 89-113, 2004,incorporated herein by reference. One of skill in the art can readilyidentify promoters of use.

The promoter can be a constitutive promoter, such as, but not limitedto, the ubiquitin promoter, see below.

The Cas9 RNA guide system consists of mature crRNA that is base-pairedto trans-activating crRNA (tracrRNA), forming a two-RNA structure thatdirects Cas9 to the locus of a desired double-stranded (ds) break intarget DNA. In some embodiments base-paired tracrRNA:crRNA combinationis engineered as a single RNA chimera to produce a guide sequence (e.g.sgRNA) which preserves the ability to direct sequence-specific Cas9dsDNA cleavage (see Jinek, M., et. al., Science. 17 Aug. 2012:337;816-821). In some embodiments, the Cas9-guide sequence complex resultsin cleavage of one or both strands at a target sequence within a gene ofinterest. Thus, the Cas9 endonuclease (Jinek, M., et. al., Science.2012; Mali, P., et. al., Nat Methods. 2013 October; 10(10): 1028-1034)and the sgRNA molecules are used sequence-specific target recognition,cleavage, and genome editing of the gene of interest. In one embodiment,the cleavage site is at a specific nucleotide, such as, but not limitedto the 16, 17, or 18th nucleotide of a 20 nucleotide target. In onenon-limiting example, the cleavage site is at the 17th nucleotide of a20-nt target sequence (see FIG. 1 and FIG. 3). The cleavage can be adouble stranded cleavage. The cleavage site can be in the coding regionof any gene, or in a non-coding region, such as in a promoter, enhancer,intron, etc. In some embodiments, a loss of function is produced. Inother embodiments, a gain of function is produced.

In some embodiments, the sgRNA molecule is selected so that the targetgenomic targets bear a protospacer adjacent motif (PAM). In someembodiments, DNA recognition by guide RNA and consequent cleavage by theendonuclease requires the presence of a protospacer adjacent motif (PAM)(e.g. 5′-NGG-3′) in immediately after the target.

In some embodiments, cleavage occurs at a site about three base-pairsupstream from the PAM. In some embodiments, the Cas9 nuclease cleaves adouble stranded nucleic acid sequence.

In some embodiments, the guide sequence is selected to reduce the degreeof secondary structure within the sequence. Secondary structure may bedetermined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold (Zuker and Stiegler, NucleicAcids Res. 9 (1981), 133-148). Another example folding algorithm is theonline webserver RNAfold, which uses the centroid structure predictionalgorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; andPA Can and GM Church, 2009, Nature Biotechnology 27(12): 1151-62). Guidesequences can be designed using the MIT CRISPR design tool found atcrispr.mit.edu or the E-CRISP tool found at www.e-crisp.org/E-CRISP.Additional tools for designing tracrRNA and guide sequences aredescribed in Naito Y et al., Bioinformatics. 2014 Nov. 20, and Ma et al.BioMed Research International, Volume 2013 (2013), Article ID 270805.The crRNA can be 18-48 nucleotides in length. The crRNA can be 18, 19,20, 21, 22, 23, 24 or 25 nucleotides in length. In one example, thecrRNA is 20 nucleotides in length. In additional embodiments, thetracrRNA is pre-optimized, and is 83 nucleotides in length, see SEQ IDNO: 3, see below:

(SEQ ID NO: 3) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

As noted above, the system disclosed herein can include a promoter, suchas, but not limited to, a U6 or H1 promoter operably linked to one ormore nucleotide sequences, such as the sgRNAs.

The U6 promoter can include the following nucleic acid sequence:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC(SEQ ID NO: 4, see also GENBANK ® Accession No.X07425.1, incorporate herein by reference).

Disclosed below is a U6 sgRNA sequence, wherein the tracrRNA isunderlined. The tracer sequence includes seven thymidines forterminating RNA transcription. The small “g,” “ga,” and the second “g”border the SapIrev and SapI sites where the nucleic acid encoding thesgRNA is inserted.

(SEQ ID NO: 5) GGCGCGCCGGATCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCgGAAGAGCgaGCTCTTCgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGGTACCGGCGCGCC

In some embodiments, more than one DNA break can be introduced by usingmore than one sgRNA. For example, two sgRNAs can be utilized, such thattwo breaks are achieved. When two or more sgRNAs are used to positiontwo or more cleavage events, in a target nucleic acid, it iscontemplated that in an embodiment the two or more cleavage events maybe made by the same or different Cas9 proteins. For example, when twosgRNAs are used to position two double strand breaks, a single Cas9nuclease may be used to create both double strand breaks.

In some embodiments, the disclosed methods include the use of one ormore vectors comprising: a) doxycycline promoter operably linked to anucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoteroperably linked to one or more nucleotide sequences encoding one or moreCRISPR-Cas guide RNAs that hybridize with the gene of interest in aeukaryotic cell. Components (a) and (b) can be located on same ordifferent vectors, whereby the one or more guide RNAs target the gene ofinterest in the eukaryotic cell and the Cas9 protein cleaves the gene ofinterest. Thus, the sequence of the gene of interest is modified in thetarget cell. Suitable vectors are disclosed above.

The disclosed methods can be used to target any gene of interest,including increasing or decreasing expression. Thus disclosed herein aremethods for the knock-in or knock-out of any gene.

Some targets, to the extent that they are present in or conditions ofthe liver are metabolic disorders, are: Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, PALB); Cirrhosis(KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988); hepatic steatosis (SIRT1,EGFR, GH, SIRT6); Cystic fibrosis (CFTR, ABCC7, CF, MRP7); Glycogenstorage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB,AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A,MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1,SCO1, HNF4a, FOXA2, FOXA1, HNF1a, FXR, LXR, PPRa, FOXO1, PGCA, PXR, CAR,RXR, NTCP, OATP, ABCA1, CX32, ABCB11), Hepatic lipase deficiency (LIPC),Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS,AXIN1, AXIN, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullarycystic kidney disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2);Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystic kidney andhepatic disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH,G19P1, PCLD, SEC63).); liver regeneration (GH, JAK2, STAT5, SHC, SOS,GRB2, RAS, RAF, MEK, ERK1/2, FAK, P130, CRKII, MEKK, JNK, P38, IRS1-3,P13K, AKT, PLC, PKC, GHR, IGF-1, IGF-2, ALS, SOCS2, SHP1, EGFR, AR, P21,HB-EGF, EGF, TGFa, C-SRC, STAT1, STAT3, P110, P85, AKT, mTOR, GSK3B,IKK, NFKB, CREB, PLC, PKC, PIP2, IP3, DAG, C-MYC, ADAM17, PDGFa, PDGFRa,PDGFRb, C/EBPa, p27), metabolic deficincies (OTC, ALB, AFP, TDO, PEPCK,UGT1A1, A1AT, TAT, ADH1, CPS), Liver detoxification (CYP2C9, CYP2C19,CYP2D6, CYP3A4, CYP3A7, CYP7A1, CYP1A2, CYP2B6, CYP2C8); Cholangiocytefunction (CFTR, SOX9, CK7, CK19, HNF6, HNF1b). Other preferred targetsinclude any one or more of include one or more of: PCSK9; Hmgcr;SERPINA1; ApoB; and.or LDL. Of course, the disclosed methods are notlimited to targeting metabolic disorders. These targets are providedonly by way of example.

In specific non-limiting embodiments, the gene of interest is SIRT1,SIRT6, SLC5A5, or β-catenin.

EXAMPLES

The disclosed methods produce iPSC or differentiated cells that undergoCRISR/Cas9 mediated recombination at a high frequency. As disclosed inFIG. 10, the efficiency of the present methods, wherein a doxycyclinepromoter operably linked to Cas9 is introduced into somatic cells beforeinduction of iPSC is 40-50%, as compared with 0.1-3% when the sameconstructs are introduced directly into iPSC.

Example 1 Generation of Tet-On-Cas9 Lentivirus A—Cas9 Validation

Two Cas9 plasmids were purchased (Addgene), one inhibiting Cas9 (Cas9i),one activating Cas9 (Cas9a). Both Cas9 sequences were PCR amplified andcloned into a validation vector pVal by recombinational cloning. NIH-3T3cells were transfected at a confluency of about 50% with the validationplasmids pVal and were incubated under standard cell culture conditionfor 48 h. Total RNA was then isolated and 1 μg was reverse transcribedusing a mixture of random hexamer and oligo-dT primer. The expression ofCas9i and Cas9a was determined by quantification of the target cDNAexpression levels relative to that found in cells transfected with theNT control vector using the vector-encoded marker transcript as internalreference gene.

B—Tet-On-Cas9 Vector Construction (FIG. 1)

The one vector lentivirus Tet-On system pcLVi(3G) (Sirion Biotech),containing a tetracycline responsive element sequence (pTRE-3G), aubiquitin C promoter (Pubq-c) and a tetracycline transactivator protein(rtTA-3G) and a puromycin resistance gene (PuroR) was used. Theconstruct is schematized in FIG. 1. To create an inducibleTet-On-Cas9i/RFP lentiviral vector, we linearized Cas9i fragment and RFPsequences and ligated them together in pcLVi(3G) vector, following thepTRE-3G sequence (Cas9i/RFP is referred as “target sequence” in FIG. 1).To create an inducible construct Tet-On-Cas9a/GFP vector, we linearizeda Cas9a fragment and GFP sequences and ligated them together inpcLVi(3G) vector, following the pTRE-3G sequence (Cas9a/GFP is referredas “target sequence” in FIG. 1). The cloning success was verified bysequencing.

C—Production of High Titer Lentivirus Stock

1×10⁶ HEK-293T cells were transfected with either the linearizedpcLVi(3G)-Tet-On-Cas9i/RFP or pcLVi(3G)-Tet-On-Cas9a-FFP lentiviralvectors to generate a high titer lentivirus production. Aftertransfection, the culture medium was harvested and the vector stockconcentrated. The biological titration of both lentivirus was performedthrough Lenti X qRT-PCR Titration kit (Clontech). This assay measuresthe number of lentivirus DNA copies integrated in the target cellgenome. Each lentivirus yields >1×10⁸ viral particles. Each stock waspreserved at −80 C.

Example 2 Transduction of Human Fetal Fibroblasts with Tet-On-Cas9Lentivirus A—Fetal Human Fibroblasts Isolation and Culture

De-identified fetal tissues were obtained with written informed consent.Human fetal fibroblasts (hFF) were isolated from fetal livers obtainedafter the termination of pregnancy performed at 20-23 weeks ofgestation. Primary hFF were isolated by digesting the tissue in EMEM(Lonza, Walkersville, Md.), which contains 0.5 mg/ml of collagenase(Type XI, SigmaAldrich, Saint-Louis Mo., Cat. #C7657), on a lab shakerfor 40 minutes. Viability was assessed by trypan blue exclusion test andwas routinely >85%. Fetal fibroblasts were plated at a density of1.3×105 cells/cm2 on type I rat tail collagen coated 12 well plates(Corning, Corning, N.Y.). Cells were cultured and passaged 2 times toget a 100% pure population of hFF, with a DMEM medium (Gibco, LifeTechnologies, Carlsbad, Calif., USA) containing 1× penstrep, 10-7M ofinsulin (Sigma-Aldrich, Saint-Louis, Mo.), and 5% bovine serum albumin(Gibco, Life Technologies, Carlsbad, Calif., USA).

B—Tet-On-Cas9 Lentiviral Transduction of Human Fetal Fibroblasts

hFF were transduced with specific lentiviral particles (Tet-On-Cas9i/RFPand Tet-On-Cas9a/GFP) at an MOI of 15. The transduced cells wereselected 72 h after transduction with 0.5 μg/mL of puromycin for 21 daysto generate a stable pool of transduced cells. Non-transduced cells diedwithin 7 days of puromycin selection (FIG. 2). After selection, totalRNA was isolated from the treated hFF. 1 μg was reverse transcribedusing a mixture of Random Hexamer and Oligo-dT primer. A qRT-PCR for thepuromycin antibiotic selection cassette was performed (FIG. 3). The twocell pools generated (hFF-Tet-On-Cas9i/RFP and hFF-Tet-On-Cas9a/GFP)were tested for absence of lentiviral particles in culture medium ofcell pools delivered with a detection of 1.0×10³ genomic copies/ml(qRT-PCR)=1.0×10¹⁰ infection units (IU)/ml (by Flow Cytometry basedassay). Quality control tests included viability, sterility (withCASO-Bouillion, Heipha) and mycoplasma testing (VENOR®GeM PCR-basedmycoplasma test, Minerva Biolabs).

C—Validation of hFF-Tet-On-Cas9 Inducible System Efficiency

To test each cell lines for Cas9 efficiency, doxycycline was added to afinal concentration of 0.5 μg/ml and cells were cultivated for 48 hours(h). The presence of fluorescent reporter proteins (RFP or GFP) wasmonitored by fluorescence microscopy (FIG. 3). Total RNA was isolatedfrom each well and 1 μg was reverse transcribed using a mixture ofRandom Hexamer and Oligo-dT primer. The expression of each Cas9 wasdetermined by quantification of the target cDNA expression levelsrelative non-induced cells and a reference gene (FIG. 5). The belowresults presented herein show reprogramming, screening andcharacterization of hiPS-TET-ON-TagRFP cells. The methods and resultswill be identical to reprogramming, screening and characterization ofhiPS-TET-ON-Cas9-GFP or hiPS-TET-ON-Cas9-RFP systems.

Example 3 Generation of Human iPS Cells Carrying an InducibleTet-On-TagRFP System

A—hFF-Tet-On-TagRFP Reprogramming into Human iPS-Tet-On-TagRFP Cells

hFF-TET-ON-TagRFP were reprogrammed into human iPS (hiPS) cells usingepisomal plasmids vectors (containing Oct3/4; Sox; Klf4; Lin; MycI andGFP) at 1 ug/mL with Lonza Nucleofactor kit (FIG. 6). The cells wereculture under mTeSR medium. After 3 weeks, more than 30 hiPS clones wereselected and were expanded separately for screening.

B—Screening for Positive hiPS-Tet-On-TagRFP Cells

To screen for positive colonies, puromycin was added to the culturemedium of each hiPS clone at a concentration of 0.125 μg/mL. hiPSnegative control cells died in 24 h whereas hiPS-Tet-On-TagRFP positivecells remained alive without any deleterious effects. Puromycin was keptin the medium for 15 days to ensure a 100% pure population of cells.hiPS-negative controls cells died within the first 24 h after puromycinaddition (FIG. 7). To test for inducible systems efficiency, doxycyclinewas added to a final concentration of 0.5 μg/ml and cells werecultivated for 48 h. The presence of RFP was monitored by fluorescencemicroscopy (FIG. 8).

Total RNA was isolated from each clone. 1 μg was reverse transcribedusing a mixture of Random Hexamer and Oligo-dT primer. A qRT-PCR for thepuromycin antibiotic selection cassette and for target genes wasperformed (FIG. 9). All hiPS clones carried the TET-ON-TagRFP systemsand 39% exhibited high levels of RFP expression (FIG. 10).

C—Characterization of hiPS-Tet-On-TagRFP Cells

Immunofluorescent staining for pluripotency-associated proteins ofNanog, Oct3/4, TRA-1-60 and SSEA4 in hiPS-Tet-On-TagRFP cell line wereperformed (FIG. 11A). Expression of pluripotency-associated genes(Oct3/4; C-myc; Lin28) was also tested by qRT-PCR expression as washiPS-TET-ON-TagRFP in cell lines (FIG. 11B).

To form embryoid bodies, cells were washed once with PBS and detachedwith Dispase for 3 minutes at 37° C. Cells were resuspended in mTeSRmedium with 20% Fetal Bovine Serum counted and plated at a concentrationof 3 million cells per ml in 6 well low attachment plates for 20 days.Cells were fixed with 4% paraformaldehyde-PBS, blocked and permeabilizedwith BSA (1%)—Triton X-100 (0.1%)—Tween (0.1%). Germ layerdifferentiation was subsequently verified using the sixfluorochrome-conjugated antibodies provided in the Human Three GermLayer 3-Color Immunocytochemistry Kit (Catalog # SCO22): fluorochromeNL557-conjugated Otx2 (red) and NL493-conjugated SOX1 (green) forEctoderm; NL557-conjugated Brachyury (red) and NL637-conjugated HAND1(green) for Mesoderm; NL637-conjugated SOX17 (red) and NL493-conjugatedGATA-4 (green) for Endoderm. All nuclei were counterstained with DAPI(blue) (FIGS. 12A, 12B).

Example 4 Functional Genome Editing and Screening with hiPS-Tet-On-Cas9with Cas9/CRISPR Technology

A schematic diagram of the technology is provided in FIG. 13.

A—Viral Production of sgRNA

A pooled plasmid library of single sgRNA (Addgene) will be transfectedinto HEK-293T cells with lentiviral packaging plasmids (Addgene). Aftertransfection, the culture medium will be harvested and the vector stockconcentrated.

B—hiPS-Tet-On-Cas9 Transduction with Lentivirus Containing sgRNA (FIG.13)

Single sgRNA or pooled genome-wide human sgRNA library lentivirus can beused. Two days before transduction, Doxycycline is added to the mediumof hiPS-Tet-On-Cas9i/RFP or hiPS-Tet-On-Cas9a/GFP cells. Both cell lines(before, during of after differentiation in any cell type) aretransduced with lentivirus. The day after, positively transduced cellsare selected, such as by adding an antibiotic selection.

C—In Vitro or In Vivo Assay of hiPS-Tet-On-Cas9 Genome Edition andScreening

Positively transduced cells are either cultured in vitro or transplantedin animal models. The functional assay evaluation depends on thescreening test characteristic (ex: proliferation in vivo; tumorformation in vivo; drug resistance in vitro etc.).

1—In Vivo Screening for Regeneration:

Non-transduced or transduced iPS-derived cells are detached bytrypsinisation and subsequently injected in the spleen of animalsconditioned for liver regeneration, namely, hepatectomy, liverradiation, drug-induced liver DNA damage, etc. three monthspost-operation, animals are sacrificed and regenerative colonies aredissected by laser capture, DNA is extracted and analyzed through nextgeneration sequencing.

2—In Vitro Screening for Drug Resistance Genes:

Positively transduced iPS-derived cells are selected by antibioticselection and cultured into 96 well plates. The cells are screened byexposure to the drug of interest. DNA is isolated from drug-resistantpopulations in each screening compared to a non-treated control groupand subjected to highthroughput sequencing analysis. In thisexperimental design gRNAs that confer survival will correspond to genesrelated to drug-resistance. This information can be used to identifymechanism for future target for disease (e.g. cancer, liver failure,regeneration, tissue preservation, etc.).

Example 5 Characterization of Cas9/CRISPR High Efficiency Activity ofhiPS-Cas9/GFP

Methods:

A) hiPS-dCas9-SAM/GFP cells were cultivated for 48 h in presence orabsence of doxycycline, expression of GFP was monitored and cells wereharvested for RNA. B) hiPS-Cas9/GFP were cultured into a single cellmedium culture (DEF-CS, Takara) and doxycycline was added for 48 h.Nucleofection was performed with plasmids carrying sgRNA for promotersof EGFR and HNF4 (sequences TGAGCTTGTTACTCGTGCCT (SEQ ID NO: 7) andGGGCGCGTTCACGCTGACCA (SEQ ID NO: 8), GenScript Cat# SC1823) or GFP ascontrol.

hiPS-Cas9/GFP were culture for 48 hours in the presence or absence ofdoxycycline. The inducible expression of GFP was monitored in 100% ofcells. RNA was harvested and the inducible expression of Cas9 system wasconfirmed (FIG. 14A).

As a proof of principle of CRISPR/Cas9 activity, a gain of function oftwo proteins normally expressed in differentiated hepatocytes, EGFR andHNF4 was tested in non-differentiated hiPS-Cas9/GFP. HNF4 is the mostcommon hepatic nuclear factor found in the liver and a marker ofspecified hepatic cells. EGFR is a transmembrane protein and a receptorfor extracellular protein ligands essential for hepatic proliferation.The expression of both proteins is required for efficient hepaticdifferentiation and proliferation. Non-differentiated hiPS-Cas9/GFPcells were culture into a single cell medium culture (DEF-CS, Takara)and doxycycline was added for 48 hours. hiPS-Cas9/GFP cells werenucleofected with two sgRNA coding for the promoters of EGFR or HNF4,purchased from GenScript SAM gRNA database, or GFP as control, at 4ug/mL with P3 Primary Cell 4D-NUCLEOFECTOR® X Kit. 36 hours afternucleofection, total RNA was isolated from each well was reversetranscribed using a mixture of Random Hexamer and Oligo-dT primer. Theexpression of endogenous EGFR and HNF4 expression was determined byRTqPCR. The results showed a drastic increase of EGFR and HNF4expression whenever the corresponding sgRNA was nucleofected in presenceof doxycycline. This result confirms the activity of hiPS-Cas9/GFP as atool for CRISPR/Cas9 high efficiency genetic engineering.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method of generating a human induced pluripotent stem cell,comprising, transfecting a human somatic cell with a nucleic acidmolecule comprising a doxycycline inducible promoter operably linked toa nucleic acid encoding a Cas9, and constitutive promoter operablylinked to a tetracycline responsive element; inducing the somatic cellto form an induced pluripotent cell, thereby producing an inducedpluripotent stem cell that can undergo CRISPR/Cas9-mediatedrecombination at a high efficiency, wherein the human inducedpluripotent cell or a cell differentiated therefrom is cultured in thepresence of doxycycline to induce expression of the Cas9.
 2. The methodof claim 1, wherein inducing the somatic cell to form an inducedpluripotent stem cell comprises transfecting the somatic cells with atleast four of: a nucleic acid molecule encoding Klf4, a nucleic acidencoding c-Myc, a nucleic acid encoding Oct4, a nucleic acid encodingSox2, a nucleic acid encoding Nanog, a nucleic acid encoding Lin28, anucleic acid encoding TRA-1-60 and SSEA4.
 3. The method of claim 2,wherein inducing the somatic cell to form an induced pluripotent stemcell comprises transfecting the somatic cells with the nucleic acidmolecule encoding Oct3/4, the nucleic acid encoding Nanog, the nucleicacid encoding TRA1-60 and the nucleic acid encoding SSEA4.
 4. The methodof claim 1, wherein the somatic cell is a fibroblast, a hepatocyte, anepithelial cell, a keratinocyte, a neuron, a myocyte, a kidney cell, alung cell, a thyroid cell, or a pancreatic cell.
 5. The method of claim1, comprising transfecting the human somatic cell with a vector encodingthe nucleic acid molecule comprising the doxycycline promoter operablylinked to the nucleic acid encoding Cas9 and the constitutive promoteroperably linked to the tetracycline responsive element.
 6. The method ofclaim 5, wherein the vector is a viral vector.
 7. The method of claim 6,wherein the viral vector is a lentiviral vector.
 8. The method of claim1, comprising transfecting the human somatic cell with a vector encodingthe nucleic acid molecule comprising the doxycycline promoter operablylinked to the nucleic acid encoding Cas9, and further comprisingintroducing a heterologous promoter operably linked to one or morenucleotide sequences encoding one or more CRISPR-Cas short guide RNAs(gRNAs) that hybridize with a gene of interest into the inducedpluripotent stem cell.
 9. The method of claim 8, wherein theheterologous promoter is a U6 promoter.
 10. The method of claim 8,wherein the guide RNAs mediate a knock-in of a desired characteristic tothe gene of interest.
 11. The method of claim 8, wherein the guide RNAsmediate a knock-out of the gene of interest.
 12. The method of claim 8,wherein the sgRNA gene targets a coding sequence for the gene ofinterest.
 13. The method of claim 8, wherein the gene of interest isSIRT1, SIRT6, SLC5A5, or β-catenin.
 14. The method of claim 1, furthercomprising differentiating the iPSC into a differentiated cell ofinterest.
 15. The method of claim 14, wherein the differentiated cell ofinterest is a fibroblast, a hepatocyte, an epithelial cell, akeratinocyte, a neuron, a myocyte, a kidney cell, a lung cell, a thyroidcell, or a pancreatic cell.
 16. The method of claim 14, wherein thedifferentiated cell is a hepatocyte.
 17. The method of claim 8, furthercomprising differentiating the induced pluripotent cell in adifferentiated cell; introducing a heterologous promoter operably linkedto one or more nucleotide sequences encoding one or more CRISPR-Casshort guide RNAs (sgRNAs) that hybridize with a gene of interest intothe differentiated cells; and culturing the differentiated cells in thepresence of doxycycline.
 18. The method of claim 1, wherein the Cas9 isCas9a or Cas9i.
 19. The method of claim 8, further comprisingdifferentiating the induced pluripotent cell in a differentiated cells.20. The method of claim 1, wherein the constitutive promoter is theubiquitin C promoter.
 21. The method of claim 1, wherein upon inducingthe somatic cells to produce the human iPSC, about 40% of the humaninduced pluripotent stem cells express the Cas9.
 22. A human inducedpluripotent stem cell (iPSC) expressing Cas9 produced by the method ofclaim 1.